Jung-Hye Kim

Akt as a Mediator of Secretory Phospholipase A2 Receptor-Involved Inducible Nitric Oxide Synthase Expression

The induction of inducible NO synthase (iNOS) by group IIA phospholipase A2 (PLA2) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA2 up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA2 receptor (sPLA2R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA2 stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA2, Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA2-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA2, and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA2-induced reporter activity of the iNOS promoter. M-type sPLA2R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA2R enhanced group IIA PLA2-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA2 catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA2 induces nitrite production by involving of M-type sPLA2R, which then mediates signal transduction events that lead to PI3K/Akt activation.

Secretory Phospholipase A2-Potentiated Inducible Nitric Oxide Synthase Expression by Macrophages Requires NF-κB Activation

The effect of secretory group II phospholipase A2 (sPLA2) on the expression of the inducible NO synthase (iNOS) and the production of NO by macrophages was investigated. sPLA2 by itself barely stimulated nitrite production and iNOS expression in Raw264.7 cells. However, in combination with LPS, the effects were synergistic. This potentiation was shown for sPLA2 enzymes from sPLA2-transfected stable cells or for purified sPLA2 from human synovial fluid. The effect of PLA2 on iNOS induction appears to be specific for the secretory type of PLA2. LPS-stimulated activation of iNOS was inhibited by the well-known selective inhibitors of sPLA2 such as 12-epi-scalaradial and ρ-bromophenacyl bromide. In contrast, the cytosolic PLA2-specific inhibitors methyl arachidonyl fluorophosphate and arachidonyltrifluoromethyl ketone did not affect LPS-induced nitrite production and iNOS expression. Moreover, when we transfected cDNA-encoding type II sPLA2, we observed that the sPLA2-transfected cells produced two times more nitrites than the empty vector or cytosolic PLA2-transfected cells. The sPLA2-potentiated iNOS expression was associated with the activation of NF-κB. We found that the NF-κB inhibitor pyrrolidinedithiocarbamate prevented nitrite production, iNOS induction, and mRNA accumulation by sPLA2 plus LPS in Raw264.7 cells. Furthermore, EMSA analysis of the activation of the NF-κB involved in iNOS induction demonstrated that pyrrolidinedithiocarbamate prevented the NF-κB binding by sPLA2 plus LPS. Our findings indicated that sPLA2, in the presence of LPS, is a potent activator of macrophages. It stimulates iNOS expression and nitrite production by a mechanism that requires the activation of NF-κB.

Effects of green tea catechin on enzyme activities and gene expression of antioxidative system in rat liver exposed to microwaves

Abstract The purpose of this study was to investigate the effect of green tea catechin on enzyme activities and gene expression of antioxidative system in rat liver exposed to microwaves. Sprague-Dawley male rats 100±10 g body weight were randomly divided into control group and microwave exposed group: Microwave exposed group was further divided into three groups: catechin free diet (MW) group, 0.25% catechin (MW-0.25C) group and 0.5% catechin (MW-0.5C) group. The rats were irradiated with microwave at frequency of 2.45 GHz for 15 min and then the changes in the pattern of antioxidative defense system and gene expression were investigated for 16 days (the 2nd, 4th, 6th, 8th and 16th days), and compared with the control group. The activity of superoxide dismutase (SOD) in MW group was lower on the 4th day after irradiation and increased in the catechin supplementation group were on the 8th day, compared with control group. The activity of glutathone peroxidase (GSHpx) in MW group was lower than that in the control group on the 8th day after irradiation, but increased to the level of the control group on the 16th day and those of MW-0.25C and MW-0.5C groups showed the same level as the control group but that was higher than the control group from 6 days after irradiation. The content of thiobarbituric acid reactive substances (TBARS) in liver of MW group was increased to 1.3, 1.5, and 1.7 fold of the control group at 2, 4, and 6 days after irradiation respectively but that in MW-0.25C and MW-0.5C groups was increased to 1.1, 1.3 and 1,3 fold of the control group at 2, 4, and 6 days but recovered to the level of the control group at 16 days after irradiation. The level of SOD gene expression in MW group was lower than that in the control group but that of MW-0.25C and MW-0.5C group were higher than the MW group. The GSHpx gene expression in MW group was expressed lower than in the control group, but expressed at a higher level in the MW-0.25C and MW-0.5C groups. It is suggested that the damage of liver tissues was alleviated and function rapidly recovered to the normal level due to probable to the correction of imbalances in the antioxidative system with the administration of green tea catechin

The effects of two new antagonists of secretory PLA2 on TNF, INOS, and COX-2 expression in activated macrophages

Phospholipase A2 (PLA2) regulates eicosanoid and platelet-activating factor production. It also plays an important role in the regulation of critical mediators in inflammatory diseases in which PLA2 activity is significantly enhanced during sepsis and multiple organ failure. Therefore, inhibitors of PLA2 activity offer themselves as target substances in the development of anti-inflammatory drugs. We identified 2 biflavonoids, bilobetin and ginkgetin, that can inhibit PLA2 activity. In experiments using 2-linol-[1-14C]PE as substrate both substances potently inhibited several kinds of type II 14-kDa PLA2 while inhibiting type I 14-kDa PLA2 to a lesser extent. We tested these PLA2 inhibitors for their ability to inhibit the production of tumor necrosis factor alpha (TNFalpha) and 2 enzymes, inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in an assay system using lipopolysaccharide (LPS)-stimulated Raw264.7 macrophages. In Raw264.7cells, bacterial LPS induced the production of COX-2 and iNOS proteins as well as TNFalpha. The inhibitors consistently inhibited the production of TNFalpha in a dose-dependent manner. Moreover, treatment of the macrophages with bilobetin and ginkgetin shut down the production of nitrite, one of the stable end products of NO released into the culture supernatant. The decrease in NO products was accompanied by a decrease in iNOS protein level as assessed by Western blot probed with specific anti-iNOS antibody. Both inhibitors also reduced the expression of COX-2 protein in the LPS-stimulated cells, which coincided with the reduction in iNOS protein. These results, therefore, suggest that these two sPLA2 inhibitors may be useful for inhibiting the production of inflammatory cytokine and NO production in inflammatory diseases.

Immediate early response of the p62 gene encoding a non-proteasomal multiubiquitin chain binding protein.

p62 is a cytoplasmic ubiquitin chain binding protein. Upon a variety of extracellular signals, both transcript and protein levels of p62 were rapidly increased. These signals include phorbol 12‐myristate 13‐acetate (PMA) and calcium ionomycin for peripheral blood mononuclear cells, serum or PDGF for serum‐starved NIH3T3 cells, IL‐3 for the G1 arrested pre‐B cell line Ba/F3, and PMA for a human promyelocyte line U937. The elevation of p62 transcript level is due to temporal stabilization of mRNA and rapid activation of the p62 gene. Cycloheximide‐induced enhanced transcription suggests the immediate early response of the p62 gene. The rapid induction of p62 indicates the presence of a novel ubiquitination‐mediated regulatory mechanism during cell proliferation and differentiation.

Group IIA secretory phospholipase A2 stimulates inducible nitric oxide synthase expression via ERK and NF-κB in macrophages

The mammalian group IIA secretory phospholipase A2 (sPLA2) is believed to play an important role in inflammation and cell injury. The present study underlines the importance of group IIA sPLA2 in the regulation of iNOS. Treatment of cells with sPLA2 induced protein expression and mRNA accumulation of iNOS in a dose‐dependent manner. The pretreatment of cells with ρ‐BPB or SCA, selective sPLA2 inhibitors, inhibited sPLA2‐induced iNOS expression. sPLA2 stimulated the simultaneous activation of two classes of mitogen‐activated protein kinases ERK and JNK, but did not stimulate p38 MAPK. PD98059, a selective MEK inhibitor, inhibited sPLA2‐induced nitrite production and iNOS expression as well as ERK phosphorylation. In addition, pretreatment of ρ‐BPB or SCA also resulted in inhibition of sPLA2‐induced ERK phosphorylation. The sPLA2 signaling mechanisms involving the activation of transcription factor NF‐κB were studied in the same cells. That stimulation of cells with sPLA2 caused NF‐κB activation in a time‐dependent manner was shown by the detection of NF‐κB‐specific DNA‐protein binding and by IκBα degradtion. sPLA2‐induced NF‐κB activation was prevented in the presence of ρ‐BPB. Furthermore, the NF‐κB inhibitor PDTC suppressed sPLA2‐induced nitrite production and iNOS expression as well as IκBα degradation. The results strongly suggest that group IIA sPLA2 induces iNOS in macrophages and that this induction occurs through ERK and NF‐κB.

Alterations of CDKN2 (MTS1/p16INK4A) gene in paraffin-embedded tumor tissues of human stomach, lung, cervix and liver cancers.

The CDKN2 (MTS1/p16INK4A) gene, encoding cyclin dependent kinase inhibitor, was found to be homozygously deleted at a high frequency in cell lines from many different types of cancer and some primary cancers. To determine the frequency of CDKN2 mutations in most common human cancers in Korea, PCR and PCR-SSCP analyses for the exon 2 of CDKN2 were performed on each set of 20 formalin-fixed and paraffin-embedded tumor tissues of stomach adenocarcinomas, lung cancers, cervix cancers and hepatocellular carcinomas. No mutations in exon 2 of CDKN2 were found in 20 stomach adenocarcinomas. In contrast to rare mutations in stomach adenocarcinomas, a high frequency of CDKN2 mutations was identified in other 3 cancers, 11 of 20 (55%) lung cancers (7 of 10 NSCLCs and 4 of 10 SCLCs), 14 of 20 (70%) cervix cancers and 11 of 20 (55%) hepatocellular carcinomas. These results suggest that mutations of the CDKN2 gene might be an important genetic change in NSCLCs, cervix cancers and hepatocellular carcinomas.

Overexpression of phospholipase Cβ-1 protects NIH3T3 cells from oxidative stress-induced cell death

Oxidative stress has been implicated in a wide range of cellular damage which includes DNA oxidation, membrane lipid peroxidation, and apoptosis. In our study, we found that overexpression of PLC-beta1 in NIH3T3 fibroblasts protected them from cell death occuring in response to oxidative stress. Cell death caused by treatment with prooxidant tert-butylhydroperoxide (TBH), H2O2, or CdCl2 was considerably suppressed in PLC-beta1 overexpressed NIH/beta1-14 cells in comparison to control NIH/neo cells. However, overexpression of PLC-beta1 failed to protect the cells from toxicity by diamide or KCN. In addition, while accumulation of c-fos mRNA was observed within 30 min of TBH treatment in vector transfected NIH/neo cells, TBH-induced c-fos mRNA generation was completely suppressed in NIH/beta1-14 cells, while that of c-jun and GAPDH was not affected. These findings suggest that PLC-beta1 may play a role in process that can protect cells from oxidative stress-induced cell death.

Implication of Egr-1 in trifluoperazine-induced growth inhibition in human U87MG glioma cells.

The early growth response gene-1 (Egr-1) is a tumor suppressor which plays an important role in cell growth, differentiation and apoptosis. Egr-1 has been shown to be down-regulated in many types of tumor tissues. Trifluoperazine (TFP), a phenothiazine class of antipsychotics, restored serum-induced Egr-1 expression in several cancer cell lines. We investigated the effect of Egr-1 expression on the TFP-induced inhibition of cell growth. Ectopic expression of Egr-1 enhanced the TFP-induced antiproliferative activity and downregulated cyclin D1 level in U87MG glioma cells. Our results suggest that antipsychotics TFP exhibits antiproliferative activity through up-regulation of Egr-1.

Methyl-beta-cyclodextrin inhibits cell growth and cell cycle arrest via a prostaglandin E(2) independent pathway.

Methyl-β-cyclodextrin, a cyclic oligosaccharide known for its interaction with the plasma membrane induces several events in cells including cell growth and anti-tumor activity. In this study, we have investigated the possible role of cyclooxygenase 2 (COX-2) in cell growth arrest induced by methyl-β-cyclodextrin in Raw264.7 macrophage cells. Methyl-β-cyclodextrin inhibited cell growth and arrested the cell cycle, and this cell cycle arrest reduced the population of cells in the S phase, and concomitantly reduced cyclin A and D expressions. Methyl-β-cyclodextrin in a dose- and time-dependent manner, also induced COX-2 expression, prostaglandin E(2) (PGE(2)) synthesis, and COX-2 promoter activity. Pretreatment of cells with NS398, a COX-2 specific inhibitor completely blocked PGE(2) synthesis induced by methyl-β-cyclodextrin, however inhibition on cell proliferation and cell cycle arrest was not effected, suggesting non-association of COX-2 in the cell cycle arrest. These results suggest that methyl-β-cyclodextrin induced cell growth inhibition and cell cycle arrest in Raw264.7 cells may be mediated by cyclin A and D1 expression.